research article perturbation of the hematopoietic profile by...

Research ArticlePerturbation of the Hematopoietic Profile byAnabolic Androgenic Steroids

Jenny Erkander Mullen1 Nina Garingrevik1 Jenny J Schulze1 Anders Rane1

Linda Bjoumlrkhem Bergman12 and Lena Ekstroumlm1

1 Division of Clinical Pharmacology Department of Laboratory Medicine Karolinska Institutet Karolinska University HospitalHuddinge 141 86 Stockholm Sweden

2Division of Clinical Microbiology Department of Laboratory Medicine Karolinska Institutet Karolinska University HospitalHuddinge 141 86 Stockholm Sweden

Correspondence should be addressed to Lena Ekstrom lenaekstromkise

Received 4 April 2014 Revised 11 September 2014 Accepted 14 September 2014 Published 22 September 2014

Academic Editor Tullio Florio

Copyright copy 2014 Jenny Erkander Mullen et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective The aim of this study was to investigate the hematopoietic profile in AAS abusers during or short after their last abuseand approximately six months later Moreover we studied if supraphysiological doses of testosterone influence the concentrationof hemoglobin and erythropoietin in healthy volunteers Design and Methods Subjects (119873 = 31) were recruited through anantidoping hotline The hematological profile was measured when the subjects entered the study and approximately 6 monthslater Testosterone enanthate (500mg) was administered to healthy volunteers (119873 = 24) Gene expression was studied in humanhek293 cells exposed to 1120583M testosterone Results Decreased levels of hemoglobin erythrocyte volume fraction and erythrocytecounts were observed after 6 months without the use of AAS Results in volunteers show that hemoglobin increased 3 four and15 days after testosterone administration whereas EPO was significantly increased by 38 four days after dose Agreeingly in vitrostudy shows that testosterone induces the mRNA level of EPO with 65 after 24-hour exposure Conclusion These results indicatethat supraphysiological doses of testosterone may cause a perturbation in the hematopoietic profileThis is of interest in relation tothe adverse cardiovascular effects observed in AAS abusers

1 Introduction

The group of anabolic-androgenic steroids (AAS) includestestosterone and its analogue synthetic derivatives and chem-ical congeners They are commonly abused by athletes andsportsmen to improve muscle mass and enhance exer-cise performance AAS are the most frequently detecteddoping agents testosterone being the predominant steroid(httpwwwwada-amaorg) Notably the abuse of theseagents among nonprofessional athletes as well as amongpeople who want to enhance their physical appearance isa growing public health problem and has become a majorsociety concern [1ndash3]

The adverse effects of AAS on the cardiovascular systeminclude adverse changes in cholesterol levels [4] endothelialdysfunction [5] and alterations of the structure of the heart

such as enlargement and thickening of the left ventriclewhich impairs its contraction and relaxation [6 7] Severalcase reports suggest that AAS may induce thrombotic eventssuch as myocardial infarction [8ndash13] A possible mechanismcontributing to thrombogenesis is an increase in hemoglobin(Hb) associated with AAS use [14] Such increases inhematocrit values have been associated with cardiovascularrisks including atherosclerosis coronary artery disease andmyocardial infarction [15ndash17]

Even though it is well known that testosterone increaseshematocrit and hemoglobin during testosterone replacementtherapy [18 19] there are few studies investigating howsupraphysiological doses of testosterone and other AAS suchas those used in doping affect the hematological system Alenfound that power athletes using AAS have higher hematocritmean corpuscular hemoglobin concentration (MCHC) and

Hindawi Publishing CorporationJournal of HormonesVolume 2014 Article ID 510257 7 pageshttpdxdoiorg1011552014510257

2 Journal of Hormones

erythrocyte sedimentation rate (ESR) compared to those nottaking AAS [20] Another small study showed that 67 ofbodybuilders using AAS had elevated hematocrit levels [21]

Whether supraphysiological doses of testosterone induceerythropoietin (EPO) production in healthy men has notbeen studied before although it is known that androgensinduce EPO concentrations in anemic patients [22 23] inrodents [24 25] and in hypogonadal men [26] The aim ofthis study was to investigate the hematological profile in AASabusers during or short after their last abuse and approx-imately six months after discontinued use Moreover westudied if supraphysiological doses of testosterone influencethe concentration of Hb and EPO in healthy volunteers

2 Subjects and Methods

21 Study Group 1 AAS Abusers Study group 1 consisted ofclinical data from an earlier study in which thirty-one menbetween 18 and 57 years old had been recruited for the projectbetween 1998 and 2002 [4] A few were recruited by referralfrom colleagues working in emergency medicine but mostwere asked to participate when contacting the antidopinghotline a free telephone counseling service for individualsaffected by or people concerned with abuse of anabolicandrogenic steroids [1] A genuine desire to give up theabuse of AAS was a prerequisite to be included Participationwas commenced after informed consent and no economicalremuneration was given to participants The project wasapproved by the Ethics Committee of the Karolinska Insti-tutet Stockholm Sweden In this study blood samples werecollected at different time points and a series of hematologicalparameters were monitored These hematological parame-ters including Hb erythrocyte counts erythrocyte volumefraction (EVF) thrombocyte count Hb mass mean corpus-cular volume (MCV) and mean corpuscular hemoglobinconcentration (MCHC) were analyzed at their first visitand compared to values six months later At each visit theparticipants met with a research nurse who could answerquestions and check their social and psychological conditionIf necessary individuals were referred to qualified medicalassessment and treatment at the psychiatric or endocrinedepartments of the hospital

22 Study Population 2 Healthy Volunteers This groupincluded 24 male volunteers aged 27ndash43 years (mean 338 plusmn47) All participants underwent a medical examinationincluding laboratory tests before enrolment All participantshad negative screening tests for illegal drugs AAS HIV andhepatitis B or C virus and were not taking any therapeuticdrugs The participants were given a 500mg intramusculardose of testosterone enanthate (Testoviron Depot) Bloodsamples were collected prior to (day 0) and four and fourteendays after testosterone administration for analyses Bloodsamples were collected between 0700 h and 0900 h afteran overnight fast The study was approved by the localethics committee and was performed in accordance with theHelsinki declarationWritten informed consent was obtainedfrom all study participants before they entered the study

23 Hematological and Hormone Measurements Hb ery-throcyte counts erythrocyte volume fraction (EVF) throm-bocyte count Hb mass mean corpuscular volume (MCV)and mean corpuscular hemoglobin concentration (MCHC)in study group 1 (AAS abusers) were analyzed at the Depart-ment of Clinical Chemistry Karolinska University Hospi-tal using standard clinical routine methods The samplesfrom study group 2 (healthy volunteers) were analyzed fortotal serum testosterone and EPO concentration also usingroutine immunochemical methods Hb was analyzed usingHemoCue analyzing system

24 Cell Experiments Hek293 cells were cultured in mini-mum essential medium 10 fetal bovine serum and 1mMsodium pyruvate All cell culture media and their ingredientswere obtained from Life Technology Cells were plated in24-well plates and grown overnight Testosterone enanthate(Sigma Aldrich) dissolved in ethanol was added to the cells(1 120583M) and grown overnight (16 h) The nontreated controlswere incubated with vehicle only Each treatment was per-formed in six independent experiments The cells were har-vested with 200120583L trizol (Invitrogen UK) and total RNAwasextracted according to manufacturerrsquos instructionsThe RNA(03 120583g) was reverse transcribed into cDNA with hexamerprimer using High Capacity cDNA Reverse Transcriptasekit (Applied Biosystems) according to the manufacturerrsquosprotocol and diluted 10 times

25 Real Time PCR ThemRNA levels of EPO in testosteronetreated Hek293 cells were determined by real time PCRGAPDH ( 4310884E Applied Biosystems) was chosen asendogenous housekeeping control gene Quantitative realtime PCR was performed using the 7500 Fast (AppliedBiosystems) Reaction mixtures contained 2xTaqman reac-tion mix (Applied Biosystems) 1 120583L of gene specific expres-sion assay (Hs01071097 m1 Applied Biosystems Foster CityCA) and 2120583L cDNA template in a total volume of 15 120583LThermal cycling conditions included activation at 95∘C(10min) followed by 45 cycles each of denaturation at95∘C (15 sec) and annealingelongation at 60∘C (1min) Eachreactionwas performed in triplicate and no-template controlswere included in each experiment The untreated sampleswere employed as calibrators and the delta delta CT-formulawas used as previously described [27] The gene expressionwas quantified as the yield of the target gene relative to thatof GAPDH gene

26 Data Analysis The concentrations of hematologicalparameters in AAS abusers and the concentration of EPOprior to and after testosterone exposure were compared usingpaired 119905-test EPO and Hb in healthy volunteers prior andafter the administration of testosterone were compared usingFriedman test followed by Dunns comparison test Themeanvalues are presented as mean plusmn SD The correlation analysisbetween EPO and total testosterone was performed usingSpearmanrsquos rank test The EPO mRNA level in Hek293 cellsprior to and after testosterone exposure was compared usingMann-Whitney test All statistical analysis was performed

Journal of Hormones 3

Table 1 Characteristic data of study group 1 at the time of inclusion(visit 1) and after 6 months cessation of AAS

Mean age (range) 264 y (18ndash57)AAS duration (range) 52 y (05ndash17)Last AAS intake (range) 84 days ago (0ndash365) Tested positive for

Nandrolone 61Testosterone 54Stanozolol 35

Total Testosterone concentrationMean (range) visit 1 191mmolL (07ndash137)Mean (range) visit 2 142mmolL (56ndash49)

Hypogonadal lt12mmolLVisit 1 63Visit 2 43

using InStat software version 501 (GraphPad San DiegoCA)

3 Results

31 Steroid Profile in AAS Abusers Thirty-one of the 56 AASsubjects that tested positive for nandrolone testosteroneandor stanozolon at the first visit did not relapse into AASabuse and returned to a 6-month follow-up were includedThe mean age was 264 (range 18ndash57) the mean time onAAS abuse was 52 years (range 05ndash17) and the mean timesince last reported AAS intake was 84 days (range 0ndash365)Fifty-four percent tested positive for testosterone 61 fornandrolone and 35 for stanozolol

Sixty-three percent of the included AAS abusers werehypogonadal (lt12mmolL total testosterone) as defined by[28] when they enrolled in the study whereas at the 6-monthfollow-up visit 43 were still identified as hypogonadal Fourof the included subjects displayed extremely high circulatoryconcentration of total testosterone (gt50mmolL) Three ofthese individuals reported a use of AAS within the last 3weeks All data about the AAS abusers study population havebeen summarized in Table 1

32 Hematological Profiles in AAS Abusers Hb was signifi-cantly higher at the first visit (mean 162 plusmn 11 gL) as com-pared to after 6 months without AAS (mean 155 plusmn 95 gL)119875 = 00005 Seventy-seven percent of the participants showedlower Hb at the second visit (Figure 1(a))

Erythrocyte count was significantly higher at the firstvisit (mean 55 plusmn 041 times 1012L) as compared to the 6-monthfollow-up (mean 52 plusmn 049) 119875 = 00008 (Figure 1(b)) Simi-larly 87 of the participants had decreased their erythrocytecount at the second visit

EVF was significantly higher at the first visit (mean 472plusmn29) than after 6 months without AAS (450 plusmn 28) 119875 =00008 (Figure 1(c)) Sixty-five of the participants exhibitlower levels at visit 2 as compared to visit 1

The level of thrombycotes Hb mass MCV and MCHCdid not significantly differ between the two visits (Figures1(d)ndash1(g))

33 Hematological Profiles in Healthy Volunteers The con-centration of EPOwas significantly 38higher four days afterthe administration of 500mg testosterone enanthate (mean1155 plusmn 341 EL) as compared to day 0 (mean 815 plusmn 266) 119875 =003 An increase was observed in 85 of the participantsThe EPO concentrations were back to baseline values on day15 (mean 1058 plusmn 430) (Figure 2(a))

There was a minor 3 increase in the Hb concentrationfour days after the testosterone injection which remained 15days after testosterone dose (119875 = 004) (Figure 2(b))

There was no correlation between the EPO and testos-terone concentrations on day 0 whereas on day 4 a significantcorrelation between the increase in EPO and increase intestosterone was observed (119903 = 064 119875 = 001) (Figure 3)

34 EPO Gene Expression in Testosterone Exposed Hek-293Cells Hek293 cells were exposed to testosterone enanthate(1 120583M) for 16 h and the mRNA levels of EPOwere determinedby real time PCR Testosterone enanthate induced EPOmRNA levels 18-fold compared to the vehicle treated controls(119875 = 001) Figure 4 These data indicate that testosteronemodifies the transcriptional activity of EPO in Hek293 cells

4 Discussion

It is generally known that AAS abuse leads to elevatedlevels of hematocrit and Hb as has been demonstrated inbodybuilders [20 21 29] Here we show for the first time thatcessation of AAS abuse results in a decrease in Hb EVF anderythrocytes count It is generally known that testosteroneaffects the hematological profile even though limited scien-tific data exists supporting this in young healthymen who aremost likely to use AAS for doping purpose Several studieshave shown that therapeutic use of testosterone stimulateserythropoiesis particularly when using testosterone esterinjections In fact erythrocytosis is the most frequent dose-limiting adverse event of testosterone replacement therapy[30] The molecular explanation behind the erythropoietinstimulatory effect of testosterone is not known but severalhypotheses propose that androgens exert a direct effect onthe bonemarrow (extensively reviewed by Shahani et al [31])Bachman et al suggested that suppression of hepcidinmay bea putative link [32]

In addition our results indicate that supraphysiologicaldoses of testosterone increase the production of EPO inhealthy volunteers Our results are in agreement with ananimal study [25] and a small human study [22] showingthat testosterone induce EPO production However somestudies failed to find an increase in EPO after administrationof testosterone in hypogonadal men [33 34] Our resultssupport the notion that testosterone induces EPO at least inhealthymen Analysis of the correlation between the increasein total testosterone andEPOonday four further supports thetheory that the increase in EPO is testosterone dependent


Hb

Visit 1 6 months later120

140

160

180

200

(gL

)

(a)

Visit 1 6 months later

Erytrocyt

4

5

6

7

(1012L)

(b)


EVF

35

40

45

50

55

60

()

(c)


Trombocyt

0

200

400

600

(109L)

(d)


Hb mass

24

26

28

30

32

34

36

(pg)

(e)


MCV

70

80

90

100

110

(fL)

(f)


MCHC

280

300

320

340

360

380

400

(gL

)

(g)

Figure 1 Hematologic profile in the AAS abusers when they entered the study (visit 1) and approximately 6 months after the cessation of theabuse The dotted line marks the minimum and maximum normal values for men between 20 and 50 years There was a significant decreasein (a) Hb (b) erythrocyte count and (c) EVF whereas there were no significant differences in (d) thrombocyte (e) Hb mass (f) MCV or (g)MCHC


25

20

15

10

5

0

EPO

(EL

)

lowastlowast

Day 0 Day 4 Day 15

(a)

180

160

140

120

Hb

(gL

)

lowast

lowast

Day 0 Day 4 Day 15

(b)

Figure 2 The concentration of (a) EPO and (b) Hb in healthy volunteers prior to the injection of 500mg testosterone enanthate (day 0) andfour and fifteen days after A significant increase in EPO was observed on day 4 but it was back to baseline on day 15 Hb was significantlyelevated on day 4 and day 15

Relative testosterone increase

Relat

ive E

PO in

crea

se

0 5 10 150

1

2

3

4

Figure 3 The relative fold increase of serum levels of testosterone(T) compared to relative increase in EPO between day 0 and day 4 Alinear regression has beenmade to fit the dataThe dotted linemarksthe 95 confidence interval of the line

175

150

125

100

075

050

025

000

Relat

ive E

PO m

RNA

expr

essio

n

lowast

Control Testosterone

Figure 4The mRNA levels in Hek293 cells exposed to testosterone(1 120583M) for 24 h There was a significant increase in EPO mRNAexpression as compared to control

at least partly This assumption received further support byour in vitro results showing that a supraphysiological dose oftestosterone increases the gene expression of EPO with 65in Hek23 cells The testosterone concentration used in ourin vitro experiments (1120583M) is within the range of the serumlevels achieved after administration of 500mg testosteroneenantahte to healthy volunteers [35] In agreement with ourfinding a recent study showed that testosterone induces themRNA expression of EPO in rats [36] It has also beenshown that androgens may increase the expression of theEPO receptor [37] indicating a further biological effect ofandrogens

We show that the EPO induction in healthy volunteerswas accompanied by an increase in Hb In contrast to EPOHb was still elevated 15 days after dose which probablyis due to the long life span of the erythrocytes [38] It ispossible that EPO and Hb may be even more elevated afterrepeated use of testosterone Even though theHb increasewasminor (intersubject variation between minus4 and +14) it maycontribute to performance enhancing effects in the athletes

The Athlete Biological Passport introduced by WADAis a new means of doping testing that aims to monitorconsecutive samples from the same individual This passportcomprises of three modules the steroid the hematocrit andthe endocrine profile [39] Our results show that the intakeof testosterone affects not only the steroid profile but also thehematocrit profile Hence an increase in Hb and EVF is causefor suspicion of AAS use just as is the case in blood dopingThis needs to be taken into account when expert panels arejudging the profiles in future test programs

Even though at least six months had passed since theAAS abusers took their last dose several of the subjects werestill displaying low levels of circulatory total testosterone attheir follow-up visit It is known that the time adapting to anormal endocrine level may take years or even be irreversiblein some cases [40ndash43] Our previous studies involving thesame AAS abusers have shown that nandrolone has a longstanding effects on LH and FSH [4] The concentrations of


total testosterone in four of the abusers were above 50 nmolLat the first visit and as high as 137 nmolL for one subjectSuch high circulatory concentrations of testosterone havebeen observed before in AAS abusers [44]

No changes in thrombocytes count were seen after dis-continued AAS use Ferenhick found that weight lifters usingAAS showed a significant trend towards increased plateletcounts compared to nonusers [45] Indirectly testosteronemay exert prothrombotic effects through its aromatizationto estradiol in contrast to nandrolone When including onlyindividuals that were positive for testosterone doping thatis with a TE ratio above 4 a significant 15 decrease inthrombocytes count was found (data not shown)

Despite an increasing knowledge that AAS use increasesthe risk of cardiovascular diseases there are as expectedno prospective interventional studies on the long termcardiovascular effects of AAS use to date [14] It is difficultto study the effects of AAS in vivo For ethical and medicalreasons only single doses of AAS may be studied in healthyvolunteers The medical examination of AAS abusers maygive some information about the effects of AAS but onedrawback using AAS users is that in some cases the AASare coused with other drugs such as clenbuterol growthhormone and aromatase inhibitors The population usedherein must be considered rather unique since the coabuse ofnarcotics was rare at the time of inclusion Only three subjectswerewithdrawn from the study due to positive narcotics testsAnother drawback with this study is that the time since theirlast AAS use differs markedly making it impossible to drawany conclusion on the time frame for normalization of thehematocrit

In conclusion we have shown that a cessation of AASuse leads to significant decrease in Hb hematocrit and ery-throcyte counts notably leading to a normalization of thesevalues In healthy men administration of supraphysiologicaldose of testosterone increased Hb and the concentrationof EPO It is possible that stimulation of erythropoiesistogether with perturbation in the cholesterol profile andendothelial health may all contribute to an increased risk ofcardiovascular events in AAS users

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by grants fromWorld Anti-DopingAgency (WADA)

References

[1] A-C Eklof A-MThurelius M Garle A Rane and F SjoqvistldquoThe anti-doping hot-line a means to capture the abuse ofdoping agents in the Swedish society and a new servicefunction in clinical pharmacologyrdquo European Journal of ClinicalPharmacology vol 59 no 8-9 pp 571ndash577 2003

[2] G Kanayama K J Brower R I Wood J I Hudson andH G Pope Jr ldquoAnabolic-androgenic steroid dependence anemerging disorderrdquo Addiction vol 104 no 12 pp 1966ndash19782009

[3] F Sjoqvist M Garle and A Rane ldquoUse of doping agentsparticularly anabolic steroids in sports and societyrdquoTheLancetvol 371 no 9627 pp 1872ndash1882 2008

[4] N Garevik E Strahm M Garle et al ldquoLong term perturbationof endocrine parameters and cholesterol metabolism after dis-continued abuse of anabolic androgenic steroidsrdquo The Journalof Steroid Biochemistry and Molecular Biology vol 127 no 3ndash5pp 295ndash300 2011

[5] C Skogastierna M Hotzen A Rane and L Ekstrom ldquoAsupraphysiological dose of testosterone induces nitric oxideproduction and oxidative stressrdquo European Journal of PreventiveCardiology vol 21 no 8 pp 1049ndash1054 2013

[6] G McKillop I C Todd and D Ballantyne ldquoIncreased leftventricular mass in a bodybuilder using anabolic steroidsrdquoBritish Journal of Sports Medicine vol 20 no 4 pp 151ndash1521986

[7] S E Campbell A Farb and K T Weber ldquoPathologic remodel-ing of themyocardium in aweightlifter taking anabolic steroidsCase reportrdquo Blood Pressure vol 2 no 3 pp 213ndash216 1993

[8] R A McNutt G S Ferenchick P C Kirlin and N J HamlinldquoAcute myocardial infarction in a 22-year-old world classweight lifter using anabolic steroidsrdquo The American Journal ofCardiology vol 62 no 1 p 164 1988

[9] M J Huie ldquoAn acute myocardial infarction occurring in ananabolic steroid userrdquo Medicine and Science in Sports andExercise vol 26 no 4 pp 408ndash413 1994

[10] R G Alvarado J Y Liu and R M Zwolak ldquoDanazol and limb-threatening arterial thrombosis two case reportsrdquo Journal ofVascular Surgery vol 34 no 6 pp 1123ndash1126 2001

[11] K McCarthy A T M Tang M J R Dalrymple-Hay andM P Haw ldquoVentricular thrombosis and systemic embolism inbodybuilders etiology and managementrdquo Annals of ThoracicSurgery vol 70 no 2 pp 658ndash660 2000

[12] T J Montine and J T Gaede ldquoMassive pulmonary embolus andanabolic steroid abuserdquo The Journal of the American MedicalAssociation vol 267 no 17 pp 2328ndash2329 1992

[13] M A Sahraian M Mottamedi A R Azimi and B MoghimildquoAndrogen-induced cerebral venous sinus thrombosis in ayoung body builder case reportrdquo BMC Neurology vol 4 no1 article 22 2004

[14] P Vanberg and D Atar ldquoAndrogenic anabolic steroid abuseand the cardiovascular systemrdquo Handbook of ExperimentalPharmacology vol 195 pp 411ndash457 2010

[15] D R Gagnon T-J Zhang F N Brand and W B KannelldquoHematocrit and the risk of cardiovascular diseasemdashthe fram-ingham study a 34-year follow-uprdquo American Heart Journalvol 127 no 3 pp 674ndash682 1994

[16] B D Spiess C Ley S C Body et al ldquoHematocrit value onintensive care unit entry influences the frequency of Q-wavemyocardial infarction after coronary artery bypass graftingrdquoThe Journal of Thoracic and Cardiovascular Surgery vol 116 no3 pp 460ndash467 1998

[17] A Goubali G Voukiklaris S Kritsikis F Viliotou and DStamatis ldquoRelation of hematocrit values to coronary heartdisease arterial hypertension and respiratory impairment inoccupational and population groups of the Athens areardquo Angi-ology vol 46 no 8 pp 719ndash725 1995


[18] S Bhasin L Woodhouse R Casaburi et al ldquoOlder men areas responsive as young men to the anabolic effects of gradeddoses of testosterone on the skeletal musclerdquo Journal of ClinicalEndocrinology andMetabolism vol 90 no 2 pp 678ndash688 2005

[19] S Bhasin LWoodhouse R Casaburi et al ldquoTestosterone dose-response relationships in healthy youngmenrdquoAmerican Journalof PhysiologymdashEndocrinology and Metabolism vol 281 no 6pp E1172ndashE1181 2001

[20] M Alen ldquoAndrogenic steroid effects on liver and red cellsrdquoBritish Journal of Sports Medicine vol 19 no 1 pp 15ndash20 1985

[21] R D Dickerman R Pertusi N Y Zachariah and F SchallerldquoAndrogen-induced erythrocytosisrdquo American Journal ofHematology vol 59 no 3 pp 263ndash264 1998

[22] N Rishpon-Meyerstein T Kilbridge J Simone and W FriedldquoThe effect of testosterone on erythropoietin levels in anemicpatientsrdquo Blood vol 31 no 4 pp 453ndash460 1968

[23] R Alexanian ldquoErythropoietin and erythropoiesis in anemicman following androgensrdquo Blood vol 33 no 4 pp 564ndash5721969

[24] D G Nathan and F H Gardner ldquoEffects of large doses ofandrogen on rodent erythropoiesis and body compositionrdquoBlood vol 26 no 4 pp 411ndash420 1965

[25] L A Malgor M Valssecia E Verges and E E de MarkowskyldquoBlockade of the in vitro effects of testosterone and erythropoi-etin on CFU-E and BFU-E proliferation by pretreatment of thedonor rats with cyproterone and flutamiderdquo Acta PhysiologicaPharmacologica etTherapeutica Latinoamericana vol 48 no 2pp 99ndash105 1998

[26] A D Coviello B Kaplan K M Lakshman T Chen A BSingh and S Bhasin ldquoEffects of graded doses of testosteroneon erythropoiesis in healthy young and older menrdquo Journal ofClinical Endocrinology and Metabolism vol 93 no 3 pp 914ndash919 2008

[27] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minus998779998779119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[28] S Arver and M Lehtihet ldquoCurrent guidelines for the diagnosisof testosterone deficiencyrdquo Frontiers of Hormone Research vol37 pp 5ndash20 2009

[29] R D Dickerman R Pertusi J Miller and N Y ZachariahldquoAndrogen-induced erythrocytosis is it erythropoietinrdquo TheAmerican Journal of Hematology vol 61 no 2 pp 154ndash155 1999

[30] S Bhasin G R Cunningham F J Hayes et al ldquoTestosteronetherapy in adult men with androgen deficiency syndromes anendocrine society clinical practice guidelinerdquo Journal of ClinicalEndocrinology and Metabolism vol 91 no 6 pp 1995ndash20102006

[31] S Shahani M Braga-Basaria M Maggio and S BasarialdquoAndrogens and erythropoiesis past and presentrdquo Journal ofEndocrinological Investigation vol 32 no 8 pp 704ndash716 2009

[32] E Bachman R Feng T Travison et al ldquoTestosterone suppresseshepcidin in men a potential mechanism for testosterone-induced erythrocytosisrdquo Journal of Clinical Endocrinology andMetabolism vol 95 no 10 pp 4743ndash4747 2010

[33] F F Ip I Di Pierro R Brown I Cunningham D J Handels-man and P Y Liu ldquoTrough serum testosterone predicts thedevelopment of polycythemia in hypogonadal men treated forup to 21 years with subcutaneous testosterone pelletsrdquo EuropeanJournal of Endocrinology vol 162 no 2 pp 385ndash390 2010

[34] M Maggio P J Snyder G P Ceda et al ldquoIs the haematopoieticeffect of testosteronemediated by erythropoietinThe results of

a clinical trial in older menrdquo Andrology vol 1 no 1 pp 24ndash282013

[35] L Ekstrom J J Schulze C Guillemette A Belanger andA Rane ldquoBioavailability of testosterone enanthate dependenton genetic variation in the phosphodiesterase 7B but not onthe uridine 51015840-diphospho- glucuronosyltransferase (UGT2B17)generdquo Pharmacogenetics and Genomics vol 21 no 6 pp 325ndash332 2011

[36] W Guo E Bachman M Li et al ldquoTestosterone administrationinhibits hepcidin transcription and is associated with increasediron incorporation into red blood cellsrdquo Aging Cell vol 12 no2 pp 280ndash291 2013

[37] V Pelekanou G Notas E Sanidas A Tsapis E Castanas andM Kampa ldquoTestosterone membrane-initiated action in breastcancer cells interaction with the androgen signaling pathwayand EPORrdquoMolecular Oncology vol 4 no 2 pp 135ndash149 2010

[38] N T Shahidi ldquoAndrogens and erythropoiesisrdquoTheNewEnglandJournal of Medicine vol 289 no 2 pp 72ndash80 1973

[39] P-E Sottas and A Vernec ldquoCurrent implementation and futureof the athlete biological passportrdquo Bioanalysis vol 4 no 13 pp1645ndash1652 2012

[40] A Clerico M Ferdeghini C Palombo et al ldquoEffect of anabolictreatment on the serum levels of gonadotropins testosteroneprolactin thyroid hormones and myoglobin of male athletesunder physical trainingrdquo The Journal of Nuclear Medicine andAllied Sciences vol 25 no 3 pp 79ndash88 1981

[41] K Boregowda L Joels J W Stephens and D E PriceldquoPersistent primary hypogonadism associated with anabolicsteroid abuserdquo Fertility and Sterility vol 96 no 1 pp e7ndashe82011

[42] J P Jarow and L I Lipshultz ldquoAnabolic steroid-inducedhypogonadotropic hypogonadismrdquo American Journal of SportsMedicine vol 18 no 4 pp 429ndash431 1990

[43] I Pirola C Cappelli A Delbarba et al ldquoAnabolic steroidspurchased on the Internet as a cause of prolonged hypogo-nadotropic hypogonadismrdquo Fertility and Sterility vol 94 no 6pp 2331ndashe1 2010

[44] G Kanayama J Kean J I Hudson and H G Pope ldquoCognitivedeficits in long-term anabolic-androgenic steroid usersrdquo Drugand Alcohol Dependence vol 130 no 1ndash3 pp 208ndash214 2013

[45] G S Ferenchick S Hirokawa E F Mammen and K ASchwartz ldquoAnabolic-androgenic steroid abuse in weight liftersevidence for activation of the hemostatic systemrdquoTheAmericanJournal of Hematology vol 49 no 4 pp 282ndash288 1995

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


erythrocyte sedimentation rate (ESR) compared to those nottaking AAS [20] Another small study showed that 67 ofbodybuilders using AAS had elevated hematocrit levels [21]

Whether supraphysiological doses of testosterone induceerythropoietin (EPO) production in healthy men has notbeen studied before although it is known that androgensinduce EPO concentrations in anemic patients [22 23] inrodents [24 25] and in hypogonadal men [26] The aim ofthis study was to investigate the hematological profile in AASabusers during or short after their last abuse and approx-imately six months after discontinued use Moreover westudied if supraphysiological doses of testosterone influencethe concentration of Hb and EPO in healthy volunteers

2 Subjects and Methods

21 Study Group 1 AAS Abusers Study group 1 consisted ofclinical data from an earlier study in which thirty-one menbetween 18 and 57 years old had been recruited for the projectbetween 1998 and 2002 [4] A few were recruited by referralfrom colleagues working in emergency medicine but mostwere asked to participate when contacting the antidopinghotline a free telephone counseling service for individualsaffected by or people concerned with abuse of anabolicandrogenic steroids [1] A genuine desire to give up theabuse of AAS was a prerequisite to be included Participationwas commenced after informed consent and no economicalremuneration was given to participants The project wasapproved by the Ethics Committee of the Karolinska Insti-tutet Stockholm Sweden In this study blood samples werecollected at different time points and a series of hematologicalparameters were monitored These hematological parame-ters including Hb erythrocyte counts erythrocyte volumefraction (EVF) thrombocyte count Hb mass mean corpus-cular volume (MCV) and mean corpuscular hemoglobinconcentration (MCHC) were analyzed at their first visitand compared to values six months later At each visit theparticipants met with a research nurse who could answerquestions and check their social and psychological conditionIf necessary individuals were referred to qualified medicalassessment and treatment at the psychiatric or endocrinedepartments of the hospital

22 Study Population 2 Healthy Volunteers This groupincluded 24 male volunteers aged 27ndash43 years (mean 338 plusmn47) All participants underwent a medical examinationincluding laboratory tests before enrolment All participantshad negative screening tests for illegal drugs AAS HIV andhepatitis B or C virus and were not taking any therapeuticdrugs The participants were given a 500mg intramusculardose of testosterone enanthate (Testoviron Depot) Bloodsamples were collected prior to (day 0) and four and fourteendays after testosterone administration for analyses Bloodsamples were collected between 0700 h and 0900 h afteran overnight fast The study was approved by the localethics committee and was performed in accordance with theHelsinki declarationWritten informed consent was obtainedfrom all study participants before they entered the study

23 Hematological and Hormone Measurements Hb ery-throcyte counts erythrocyte volume fraction (EVF) throm-bocyte count Hb mass mean corpuscular volume (MCV)and mean corpuscular hemoglobin concentration (MCHC)in study group 1 (AAS abusers) were analyzed at the Depart-ment of Clinical Chemistry Karolinska University Hospi-tal using standard clinical routine methods The samplesfrom study group 2 (healthy volunteers) were analyzed fortotal serum testosterone and EPO concentration also usingroutine immunochemical methods Hb was analyzed usingHemoCue analyzing system

24 Cell Experiments Hek293 cells were cultured in mini-mum essential medium 10 fetal bovine serum and 1mMsodium pyruvate All cell culture media and their ingredientswere obtained from Life Technology Cells were plated in24-well plates and grown overnight Testosterone enanthate(Sigma Aldrich) dissolved in ethanol was added to the cells(1 120583M) and grown overnight (16 h) The nontreated controlswere incubated with vehicle only Each treatment was per-formed in six independent experiments The cells were har-vested with 200120583L trizol (Invitrogen UK) and total RNAwasextracted according to manufacturerrsquos instructionsThe RNA(03 120583g) was reverse transcribed into cDNA with hexamerprimer using High Capacity cDNA Reverse Transcriptasekit (Applied Biosystems) according to the manufacturerrsquosprotocol and diluted 10 times

25 Real Time PCR ThemRNA levels of EPO in testosteronetreated Hek293 cells were determined by real time PCRGAPDH ( 4310884E Applied Biosystems) was chosen asendogenous housekeeping control gene Quantitative realtime PCR was performed using the 7500 Fast (AppliedBiosystems) Reaction mixtures contained 2xTaqman reac-tion mix (Applied Biosystems) 1 120583L of gene specific expres-sion assay (Hs01071097 m1 Applied Biosystems Foster CityCA) and 2120583L cDNA template in a total volume of 15 120583LThermal cycling conditions included activation at 95∘C(10min) followed by 45 cycles each of denaturation at95∘C (15 sec) and annealingelongation at 60∘C (1min) Eachreactionwas performed in triplicate and no-template controlswere included in each experiment The untreated sampleswere employed as calibrators and the delta delta CT-formulawas used as previously described [27] The gene expressionwas quantified as the yield of the target gene relative to thatof GAPDH gene

26 Data Analysis The concentrations of hematologicalparameters in AAS abusers and the concentration of EPOprior to and after testosterone exposure were compared usingpaired 119905-test EPO and Hb in healthy volunteers prior andafter the administration of testosterone were compared usingFriedman test followed by Dunns comparison test Themeanvalues are presented as mean plusmn SD The correlation analysisbetween EPO and total testosterone was performed usingSpearmanrsquos rank test The EPO mRNA level in Hek293 cellsprior to and after testosterone exposure was compared usingMann-Whitney test All statistical analysis was performed








3 Results











4 Discussion




Hb


140

160

180

200

(gL

)

(a)


Erytrocyt

4

5

6

7

(1012L)

(b)


EVF

35

40

45

50

55

60

()

(c)


Trombocyt

0

200

400

600

(109L)

(d)


Hb mass

24

26

28

30

32

34

36

(pg)

(e)


MCV

70

80

90

100

110

(fL)

(f)


MCHC

280

300

320

340

360

380

400

(gL

)

(g)



25

20

15

10

5

0

EPO

(EL

)

lowastlowast

Day 0 Day 4 Day 15

(a)

180

160

140

120

Hb

(gL

)

lowast

lowast

Day 0 Day 4 Day 15

(b)



Relat

ive E

PO in

crea

se

0 5 10 150

1

2

3

4


175

150

125

100

075

050

025

000

Relat

ive E

PO m

RNA

expr

essio

n

lowast














Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results











4 Discussion




Hb


140

160

180

200

(gL

)

(a)


Erytrocyt

4

5

6

7

(1012L)

(b)


EVF

35

40

45

50

55

60

()

(c)


Trombocyt

0

200

400

600

(109L)

(d)


Hb mass

24

26

28

30

32

34

36

(pg)

(e)


MCV

70

80

90

100

110

(fL)

(f)


MCHC

280

300

320

340

360

380

400

(gL

)

(g)



25

20

15

10

5

0

EPO

(EL

)

lowastlowast

Day 0 Day 4 Day 15

(a)

180

160

140

120

Hb

(gL

)

lowast

lowast

Day 0 Day 4 Day 15

(b)



Relat

ive E

PO in

crea

se

0 5 10 150

1

2

3

4


175

150

125

100

075

050

025

000

Relat

ive E

PO m

RNA

expr

essio

n

lowast














Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hb


140

160

180

200

(gL

)

(a)


Erytrocyt

4

5

6

7

(1012L)

(b)


EVF

35

40

45

50

55

60

()

(c)


Trombocyt

0

200

400

600

(109L)

(d)


Hb mass

24

26

28

30

32

34

36

(pg)

(e)


MCV

70

80

90

100

110

(fL)

(f)


MCHC

280

300

320

340

360

380

400

(gL

)

(g)



25

20

15

10

5

0

EPO

(EL

)

lowastlowast

Day 0 Day 4 Day 15

(a)

180

160

140

120

Hb

(gL

)

lowast

lowast

Day 0 Day 4 Day 15

(b)



Relat

ive E

PO in

crea

se

0 5 10 150

1

2

3

4


175

150

125

100

075

050

025

000

Relat

ive E

PO m

RNA

expr

essio

n

lowast














Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25

20

15

10

5

0

EPO

(EL

)

lowastlowast

Day 0 Day 4 Day 15

(a)

180

160

140

120

Hb

(gL

)

lowast

lowast

Day 0 Day 4 Day 15

(b)



Relat

ive E

PO in

crea

se

0 5 10 150

1

2

3

4


175

150

125

100

075

050

025

000

Relat

ive E

PO m

RNA

expr

essio

n

lowast














Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article perturbation of the hematopoietic profile by...

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